Protocol for AI-assisted quantitative analysis and setup of tumor spheroid invasion into tissue.

Recent work has revealed an active mechanical role for the tissue barrier in collective invasion, offering a new perspective on tissue invasion and morphogenesis. Here, we present a protocol for AI-assisted quantitative analysis and setup of tumor spheroid invasion into mesothelial tissue. We describe steps for co-culture setup, time-lapse imaging, AI training, and segmentation. We then detail procedures for cell tracking and quantitative analysis to study the fracturing process of the mesothelium. For complete details on the use and execution of this protocol, please refer to Wu et al.1.